TY - JOUR
T1 - Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation
AU - Gilbert, Luke A.
AU - Horlbeck, Max A.
AU - Adamson, Britt
AU - Villalta, Jacqueline E.
AU - Chen, Yuwen
AU - Whitehead, Evan H.
AU - Guimaraes, Carla
AU - Panning, Barbara
AU - Ploegh, Hidde L.
AU - Bassik, Michael C.
AU - Qi, Lei S.
AU - Kampmann, Martin
AU - Weissman, Jonathan S.
N1 - Funding Information:
The authors thank M. Tanenbaum, J. Tsai, K. Kostova, J. Zalatan, W. Lim, S. Weissman, J. Doudna, and R. Vale for unpublished reagents, technical advice and helpful discussion. L.S.Q. acknowledges support from the UCSF Center for Systems and Synthetic Biology. This work was supported by NIH P50 GM102706 (J.S.W.), NIH P50 GM081879 (L.S.Q., E.H.W.), NIH U01 CA168370 and NIH R01 DA036858 (J.S.W.), as well as the Howard Hughes Medical Institute (J.E.V., Y.C., M.K., M.A.H., J.S.W.). B.A. is an HHMI Fellow of the Damon Runyon Cancer Research Foundation (DRG-[2182-14]). L.A.G. is a Fellow of the Leukemia and Lymphoma Society. M.A.H. is supported by the UCSF Medical Scientist Training Program. M.K. is supported by NCI/NIH Pathway to Independence Award K99CA181494.
Publisher Copyright:
© 2014 Elsevier B.V. All rights reserved.
PY - 2014/10/23
Y1 - 2014/10/23
N2 - While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.
AB - While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.
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U2 - 10.1016/j.cell.2014.09.029
DO - 10.1016/j.cell.2014.09.029
M3 - Article
C2 - 25307932
AN - SCOPUS:84908352138
SN - 0092-8674
VL - 159
SP - 647
EP - 661
JO - Cell
JF - Cell
IS - 3
ER -